Synthetic cannabinoids (SCBs) are a class of designer drugs comprising more than 500 distinct compounds in thirteen different structurally diverse families, and are widely abused as an alternative to cannabis. The effects of consuming SCBs include adverse psychological and physiological effects and even death. SCB overdose is currently diagnosed in emergency room (ER) settings from indirect evidence, including self-reporting or the presence of both a cannabimimetic toxidrome and a negative drug screen. Therefore, there is a need for accurate screening methods for SCBs/metabolites in serum that can be employed in the ER, to diagnose and distinguish SCB overdoses from other psychiatric or neurological diseases for establishing appropriate treatment protocols and disposition measures. Immunoassays have been developed for certain SCBs. These assays rely on specific binding between drugs/metabolites and antibodies, offering high specificity and sensitivity. However, minor modifications are often introduced to the SCB core structure to evade regulation. These modifications can greatly impair binding by existing antibodies, resulting in false-negative results. With hundreds of SCBs on the market and new compounds continuously emerging, it is therefore urgent to develop new cross-reactive bioaffinity elements that can recognize structurally similar SCBs. To address this problem, an original homogeneous nuclease-assisted (NA)-SELEX in conjunction with a parallel-and-serial selection strategy is proposed to isolate a high-affinity DNA aptamer that cross reacts to all SCBs and their metabolites from the indazole-3-carboxamide family. NA-SELEX utilizes a structured DNA library and a high-fidelity restriction enzyme to efficiently separate target-bound aptamers from the remainder of the library. Specifically, target binding causes aptamers to undergo a conformational change that renders them inaccessible to enzymatic digestion, whereas unbound oligonucleotides are viable enzyme substrates and are rapidly digested and eliminated from the next round of selection. Counter-SELEX will be performed to ensure that the isolated aptamer does not bind to interferent molecules including phytocannabinoids, structurally-similar endogenous substances, prescription drugs, illicit drugs as well as structurally-dissimilar drugs associated with ER visits. This process is expected to yield the first cross-reactive aptamer capable of recognizing numerous SCBs and metabolites based on their shared core structure, such that peripheral chemical modifications should not meaningfully affect the aptamer's binding affinity. An electrochemical aptamer-based sensor based on this cross-reactive aptamer will then be fabricated to sense the total serum concentration of all parent SCBs and their metabolites, achieving a clinically relevant detection limit and long detection window. The resulting sensor can be used in the ER to aid in the diagnosis of SCB overdose, greatly improving public health and safety. The proposed technologies can easily be adapted to isolate high-affinity, cross-reactive aptamers for other families of novel psychoactive substances or for in vivo therapeutic and diagnostic applications.